The long-term goal of our research is to understand and manipulate the antigen processing mechanisms that yields thousands of peptide/MHC class I complexes on the cell surface. Contrary to the text book model which depicts the antigenic peptides being generated solely in the cytoplasm, recent findings indicate that antigen processing occurs both in the cytoplasm and continues in the secretory pathway specifically the endoplasmic reticulum (ER). The protease that mediates the trimming of antigenic peptides has recently been identified and termed ERAAP, for an ER aminopeptidase associated with antigen processing. Here we propose to fill the gaps in our understanding of how peptide trimming occurs in the ER and its impact on the CD8 T cell repertoire in a physiological context. We will use the unique tools we have developed in our laboratory for this analysis. These include (a) the ERAAP knock-out mice, and (b) novel methods for analysis of final output of the antigen processing pathway as well as the elusive proteolytic intermediates that are generated in normal and professional antigen presenting cells.